Whole Blood Assay Protocol

							Whole Blood Assay Protocol
General:
     Blood drawn into plasma/EDTA tubes
     Sterile technique required
     Culture media: RPMI + 2nM L-glutamine
Dilutions for LPS: Stock = 500mg/ml (Final concentration 100ng/ml)

Tube A) 4l from stock + 36 l media = 40l (50g/ml LPS)
Tube B) 30l from tube A + 270 l media=300l (5g/ml LPS)
Tube C) 250l from tube B + 2250 l media= 2.5 ml (500ng/ml LPS)
On plate dilution 1 part of LPS: 4 parts media = 100ng/ml LPS
Assay
   1. Materials must be prepared in advance of drawing blood. Results will suffer if
      more than 30 minutes from bleeding to plating.
   2. Obtain at least 2 ml of patient blood in EDTA vacutainer tube.
   3. Use 96-wells round bottom plates
   4. Plate A) LPS (+): with a multichannel pipettor add 70 l of 37oC culture media, +
      20l from LPS tube C + 10l of whole blood.
      Plate B) LPS (-) add 90 l of 37oC culture media + 10l of whole blood.
   5. Incubate overnight at 37oC, 5 % CO2
   6. Do not spin plate prior to harvest
   7. Collect 40l supernatant from each well and aliquot in corresponding position of
      96 wells non-binding plates,
   8. Freeze at –80oC until use.
   Considerations
   Gently invert vacutainer prior to plating blood, cells will settle out otherwise.
   Gently mix blood/media/LPS mixture in reservoir while pippeting.
   Suspension will be viscous when harvesting supernatants, be sure to aspirate
   identical amounts in each channel of the pipettor.